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2.
Nat Commun ; 11(1): 4625, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934225

RESUMO

A hallmark of neurodegeneration is defective protein quality control. The E3 ligase Listerin (LTN1/Ltn1) acts in a specialized protein quality control pathway-Ribosome-associated Quality Control (RQC)-by mediating proteolytic targeting of incomplete polypeptides produced by ribosome stalling, and Ltn1 mutation leads to neurodegeneration in mice. Whether neurodegeneration results from defective RQC and whether defective RQC contributes to human disease have remained unknown. Here we show that three independently-generated mouse models with mutations in a different component of the RQC complex, NEMF/Rqc2, develop progressive motor neuron degeneration. Equivalent mutations in yeast Rqc2 selectively interfere with its ability to modify aberrant translation products with C-terminal tails which assist with RQC-mediated protein degradation, suggesting a pathomechanism. Finally, we identify NEMF mutations expected to interfere with function in patients from seven families presenting juvenile neuromuscular disease. These uncover NEMF's role in translational homeostasis in the nervous system and implicate RQC dysfunction in causing neurodegeneration.


Assuntos
Doenças Neuromusculares/metabolismo , Ribossomos/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Doenças Neuromusculares/genética , Doenças Neuromusculares/patologia , Proteólise , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência
4.
Acta Neuropathol ; 135(3): 459-474, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29196813

RESUMO

Hexanucleotide repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (C9 ALS). The main hypothesized pathogenic mechanisms are C9orf72 haploinsufficiency and/or toxicity from one or more of bi-directionally transcribed repeat RNAs and their dipeptide repeat proteins (DPRs) poly-GP, poly-GA, poly-GR, poly-PR and poly-PA. Recently, nuclear import and/or export defects especially caused by arginine-containing poly-GR or poly-PR have been proposed as significant contributors to pathogenesis based on disease models. We quantitatively studied and compared DPRs, nuclear pore proteins and C9orf72 protein in clinically related and clinically unrelated regions of the central nervous system, and compared them to phosphorylated TDP-43 (pTDP-43), the hallmark protein of ALS. Of the five DPRs, only poly-GR was significantly abundant in clinically related areas compared to unrelated areas (p < 0.001), and formed dendritic-like aggregates in the motor cortex that co-localized with pTDP-43 (p < 0.0001). While most poly-GR dendritic inclusions were pTDP-43 positive, only 4% of pTDP-43 dendritic inclusions were poly-GR positive. Staining for arginine-containing poly-GR and poly-PR in nuclei of neurons produced signals that were not specific to C9 ALS. We could not detect significant differences of nuclear markers RanGap, Lamin B1, and Importin ß1 in C9 ALS, although we observed subtle nuclear changes in ALS, both C9 and non-C9, compared to control. The C9orf72 protein itself was diffusely expressed in cytoplasm of large neurons and glia, and nearly 50% reduced, in both clinically related frontal cortex and unrelated occipital cortex, but not in cerebellum. In summary, sense-encoded poly-GR DPR was unique, and localized to dendrites and pTDP43 in motor regions of C9 ALS CNS. This is consistent with new emerging ideas about TDP-43 functions in dendrites.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Encéfalo/metabolismo , Proteína C9orf72/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dipeptídeos/metabolismo , Medula Espinal/metabolismo , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Encéfalo/patologia , Proteína C9orf72/genética , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Citoplasma/metabolismo , Citoplasma/patologia , Expansão das Repetições de DNA , Dendritos/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neuroglia/metabolismo , Neuroglia/patologia , Medula Espinal/patologia
5.
Neuron ; 90(3): 535-50, 2016 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-27112497

RESUMO

Hexanucleotide expansions in C9ORF72 are the most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Disease mechanisms were evaluated in mice expressing C9ORF72 RNAs with up to 450 GGGGCC repeats or with one or both C9orf72 alleles inactivated. Chronic 50% reduction of C9ORF72 did not provoke disease, while its absence produced splenomegaly, enlarged lymph nodes, and mild social interaction deficits, but not motor dysfunction. Hexanucleotide expansions caused age-, repeat-length-, and expression-level-dependent accumulation of RNA foci and dipeptide-repeat proteins synthesized by AUG-independent translation, accompanied by loss of hippocampal neurons, increased anxiety, and impaired cognitive function. Single-dose injection of antisense oligonucleotides (ASOs) that target repeat-containing RNAs but preserve levels of mRNAs encoding C9ORF72 produced sustained reductions in RNA foci and dipeptide-repeat proteins, and ameliorated behavioral deficits. These efforts identify gain of toxicity as a central disease mechanism caused by repeat-expanded C9ORF72 and establish the feasibility of ASO-mediated therapy.


Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Demência Frontotemporal/tratamento farmacológico , Fatores de Troca do Nucleotídeo Guanina/genética , Oligonucleotídeos Antissenso/farmacologia , RNA/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Proteína C9orf72 , Expansão das Repetições de DNA/genética , Demência Frontotemporal/genética , Camundongos Transgênicos , Neurônios/metabolismo , Oligonucleotídeos Antissenso/efeitos adversos , Oligonucleotídeos Antissenso/genética
6.
Neurol Clin ; 33(4): 855-76, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26515626

RESUMO

The neuropathologic molecular signature common to almost all sporadic amyotrophic lateral sclerosis (ALS) and most familial ALS is TDP-43 immunoreactive neuronal cytoplasmic inclusions. The neuropathologic and molecular neuropathologic features of ALS variants, primarily lateral sclerosis and progressive muscular atrophy, are less certain but also seem to share the primary features of ALS. Genetic causes, including mutations in SOD1, TDP-43, FUS, and C9orf72, all have distinctive molecular neuropathologic signatures. Neuropathology will continue to play an increasingly key role in solving the puzzle of ALS pathogenesis.


Assuntos
Esclerose Lateral Amiotrófica , Proteínas de Ligação a DNA/genética , Variação Genética/genética , Proteínas/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/fisiopatologia , Proteína C9orf72 , Humanos
7.
Genetics ; 193(2): 421-30, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23150604

RESUMO

Site-specific recombinases (SSRs) are valuable tools for manipulating genomes. In Drosophila, thousands of transgenic insertions carrying SSR recognition sites have been distributed throughout the genome by several large-scale projects. Here we describe a method with the potential to use these insertions to make custom alterations to the Drosophila genome in vivo. Specifically, by employing recombineering techniques and a dual recombinase-mediated cassette exchange strategy based on the phiC31 integrase and FLP recombinase, we show that a large genomic segment that lies between two SSR recognition-site insertions can be "captured" as a target cassette and exchanged for a sequence that was engineered in bacterial cells. We demonstrate this approach by targeting a 50-kb segment spanning the tsh gene, replacing the existing segment with corresponding recombineered sequences through simple and efficient manipulations. Given the high density of SSR recognition-site insertions in Drosophila, our method affords a straightforward and highly efficient approach to explore gene function in situ for a substantial portion of the Drosophila genome.


Assuntos
Drosophila melanogaster/genética , Marcação de Genes , Genoma de Inseto , Mutagênese Insercional , Animais , DNA Nucleotidiltransferases/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Integrases/genética , Mutagênese Sítio-Dirigida , Proteínas Repressoras/genética
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